Altino Choupina

Transglutaminases : recent achievements and new sources

Transglutaminases are a family of enzymes (EC 2.3.2.13), widely distributed in various organs, tissues, and body fluids, that catalyze the formation of a covalent bond between a free amine group and the γ-carboxamide group of protein or peptide-bound glutamine. Besides forming these bonds, that exhibit high resistance to proteolytic degradation, transglutaminases also form extensively cross-linked, generally insoluble, protein biopolymers that are indispensable for the organism to create barriers and stable structures. The extremely high cost of transglutaminase of animal origin has hampered its wider application and has initiated efforts to find an enzyme of microbial origin. Since the early 1990s, many microbial transglutaminase-producing strains have been found, and production processes have been optimized. This has resulted in a rapidly increasing number of applications of transglutaminase in the food sector. However, applications of microbial transglutaminase in other sectors have also been explored, but in a much lesser extent. Our group has identified a transglutaminase in the oomycete Phytophthora cinnamomi, which is able to induct defense responses and disease-like symptoms. In this mini-review, we report the achievements in this area in order to illustrate the importance and the versatility of transglutaminases.

Developments in the Fermentation Process and Quality Improvement Strategies for Mead Production

Mead is a traditional alcoholic drink derived from the fermentation of diluted honey in the presence of appropriate yeast. Its modern production, in general terms, involves the addition of nutrients to initial diluted honey, pasteurization, yeast inoculation, fermentation and removal of impurities. Undesirable events along the process have been reported; among them, we highlight: delayed or arrested fermentations, modified and unpleasant sensory and quality parameters of the final product. These problems have been linked to the inability of yeasts to accomplish their role in extreme growth conditions. Emphasis has also been placed on the long fermentation times required, ranging from weeks to months, particularly when traditional procedures are applied and when the honey concentration is low. A series of alterations to the must and technological changes have been proposed in order to optimize the mead production process. In this context, this review examines the evidence that aims to improve meads’ quality and make the production process easier and more efficient, by clarifying the source of unexpected events, describing the implementation of different fermentative microorganisms and using new methodologies.

Scientifically advanced solutions for chestnut ink disease

On the north regions of Portugal and Spain, the Castanea sativa Mill. culture is extremely important. The biggest productivity and yield break occurs due to the ink disease, the causal agent being the oomycete Phytophthora cinnamomi. This oomycete is also responsible for the decline of many other plant species in Europe and worldwide. P. cinnamomi and Phytophthora cambivora are considered, by the generality of the authors, as the C. sativa ink disease causal agents. Most Phytophthora species secrete large amounts of elicitins, a group of unique highly conserved proteins that are able to induce hypersensitive response (HR) and enhances plant defense responses in a systemic acquired resistance (SAR) manner against infection by different pathogens. Some other proteins involved in mechanisms of infection by P. cinnamomi were identified by our group: endo-1,3-beta-glucanase (complete cds); exo-glucanase (partial cds) responsible by adhesion, penetration, and colonization of host tissues; glucanase inhibitor protein (GIP) (complete cds) responsible by the suppression of host defense responses; necrosis-inducing Phytophthora protein 1 (NPP1) (partial cds); and transglutaminase (partial cds) which inducts defense responses and disease-like symptoms. In this mini-review, we present some scientifically advanced solutions that can contribute to the resolution of ink disease.

Isolation and Characterization by Asymmetric PCR of the ENDO1 Gene for Glucan endo-1,3-β-D-glucosidase in Phytophthora cinnamomi Associated with the ink Disease of Castanea sativa Mill.

Ink disease is one of the most destructive diseases in Castanea sativa . The most common symptoms are root necrosies and a reduction in root growth, which inv ariably lead to the death of the trees. Phytophthora cinnamomi is an oomycete associated with this disease whose life cycle deve lops integrally in the soil. In the present work, w as a fragment with 1231bp of the glucan endo-1,3- β-D-glucosidase gene obtained by amplification, usin g conserved primers and the full-length gene sequence by flanki ng this known sequence by asymmetric PCR. This frag ment was obtained from genomic DNA of Phytophthora cinnamomi isolated in the European Regions of Castilla-Leon (Spain) and Tras-os-Montes (Portugal) and associated with t he ink disease of Castanea sativa Mill.

Isolation and Sequencing of Actin1, Actin2 and Tubulin1 Genes Involved in Cytoskeleton Formation in Phytophthora cinnamomi

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. On the Nordeste Transmontano region (northeast Portugal), the Castanea sativa chestnut culture is extremely important. The biggest productivity and yield break occurs due to the ink disease, caused by Phytophthora cinnamomi which is one of the most widely distributed Phytophthora species, with nearly 1000 host species. The knowledge about molecular mechanisms responsible for pathogenicity is an important tool in order to combat associate diseases of this pathogen. Complete open reading frames (ORFs) of act1, act2 and tub1 genes who participate in cytoskeleton formation in P. cinnamomi were achieved by high-efficiency thermal asymmetric interlaced (HE-TAIL) polymerase chain reaction (PCR). act1 gene comprises a 1128 bp ORF, encoding a deduced protein of 375 amino acids (aa) and 41,972 kDa. act2 ORF comprises 1083 bp and encodes a deduced protein of 360 aa and 40,237 kDa. tub1 has a total length of 2263 bp and encodes a 453 aa protein with a molecular weight of 49.911 kDa. Bioinformatics analyses shows that actin1 is ortholog to the act1 genes of Phytophthora infestans, Phytophthora megasperma and Phytophthora melonis; actin2 is ortholog to the act2 genes of P. infestans, Phytophthora brassicae, P. melonis and Pythium splendens and tubulin1 shows the highest orthology to P. infestans and P. capsici α-tubulin genes. Analysed 3D structure of the three putative proteins revealed a spatial conformation highly similar to those described for orthologous proteins obtained by X-ray diffraction.

Protein-Glutamine Gamma-Glutamyltransferase

Transglutaminases (TGs) are a family of enzymes (EC 2.3.2.13) that catalyze the formation of amide bonds between proteins to form insoluble crosslinked protein aggregates that are resistant to chemicals, detergents, and proteases degradation (Griffin et al. 2002; Yokoyama et al. 2004). TG activity was first observed in 1957 by Clarke et al. (1957), when they found an enzyme with transamidating properties extracted from guinea pig liver. Only in 1959 the name transglutaminase was assigned by Waelsch and collaborators (Mycek et al. 1959), in order to distinguish this enzymatic activity from that of other enzymes with similar activity. TGs catalyzes posttranslational cross-link reactions between two substrates that can be two proteins or two residues of the same protein, involving lysine and glutamine. More specifically, TGs catalyze acyl-transfer reactions introducing covalent crosslinks between a g-carboxyamide group of glutamine residue and e-amino group of lysine residue or other primary amines. Peptides and various primary amines act as acyl donors, and primary amino groups (including e-amino groups of lysine residues), either as Protein-Glutamine Gamma-Glutamyltransferase 4253

Genes found in partial sequencing od Phytophthora cinnamomi

The Project COMBATINTA/SP2.P11/02 Interreg IIIA – Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Found, supported this work.

A extrusão em tecnologia alimentar: aplicações, caraterísticas dos produtos, composição e tendências futuras

Food extrusion involves complex molecular transformations that allow diversity in the existing extruded products and in their physical, chemical, sensory and nutritional properties. In this article, current applications of extrusion in food industry are presented, as well as the characteristics of the extruded products; it also describes the interactions between the relevant parameters involved in this particular food processing operation. Finally, future trends in the extruded products formulation are presented.